Phosphoinositides (PIPns), e.g., phosphatidylinositol 4,5-bisphosphate (PI 4,5) P2) are key signaling molecules in cellular communication. Specific PIPns recruit target signaling proteins and intracellular adapter proteins to inner membrane sites to activate protein kinases and thereby initiate signal transduction cascades in order to regulate exo- and endocytosis, protein sorting, cell proliferation and migration, tumorigenesis, apoptosis, and the control of cell shape. Determining the selectivity of a given target protein for a specific PIPn has become an important issue in deciphering downstream effectors and in new drug discovery efforts for potential therapeutics that could control protein-PIPn interactions. We propose to develop both components of an efficient protein overlay system that allows determining lipid selectivity of a known protein or levels of a given PIPn produced by lipid kinase and phosphatase reactions. Both methods use the same principle, i.e., detection of proteins bound to PIPns immobilized on nitrocellulose. First, we will optimize the lipid blot method, which we call "PIP-Strips (TM)." Second, we will develop highly selective, high-affinity proteins that recognize a specific PIPn using two coupled pleckstrin homology (PH) or FYVE domains; this new strategy is called Double PH(tm). The reagents prepared and optimized in this Phase I project will be used in Phase II to develop a variety of high throughput screening methods, e.g., rapid, automated assays for PIPn kinases and phosphatase and screening for small molecule inhibitors of protein-PIPn binding PROPOSED COMMERCIAL APPLICATION: A number of commercial applications have been envisioned for the products developed in this grant. PIP-Strips(TM) will be primarily sold as tools to determine lipid binding specificity of isolated proteins, their original purpose. Other applications include antibody screening and in house quality control of cloned proteins. Lipo-PIP-Strips(TM) will also fill these same roles. The Double-PH reagents will be sold as individual items targeted to a specific lipid and a kits focussing on a class of lipids such as 3'-phosphorylated PIPns.